Specific mutations in H5N1 mainly impact the magnitude and velocity of the host response in mice

Tchitchek, Nicolas; Eisfeld, Amie J; Tisoncik-Go, Jennifer; Josset, Laurence; Gralinski, Lisa E; Bécavin, Christophe; Tilton, Susan C; Webb-Robertson, Bobbie-Jo; Ferris, Martin T; Totura, Allison L; Li, Chengjun; Neumann, Gabriele; Metz, Thomas O; Smith, Richard D; Waters, Katrina M; Baric, Ralph; Kawaoka, Yoshihiro; Katze, Michael G

An AMT tag database of identified peptides was developed for virus-infected lungs, using mock-infected and virus-infected samples from two studies: 1) H5N1 data reported here and 2) another with SARS-CoV MA15. The details of study 2 will be described elsewhere. To generate the AMT tag database, aliquots of the H5N1or mock-infected samples were combined to make the following pools: 1) mock-infection (all time points), 2) early infection (1 and 2 d), and 3) late infection (4 and 7 d). Samples from the SARS-CoV MA15 studies were similarly pooled. Each pool was subjected to strong cation exchange fractionation as described above, and each fraction was analyzed by nanocapillary LC-MS/MS. The SEQUEST analysis software [56] was used to match the MS/MS fragmentation spectra with sequences from the April 20, 2010 UniProt/Swiss-Prot rodent protein database, containing 16,244 entries (protein TITIN_Mouse was removed due to excessive length). The data were also matched to sequences from the H5N1 viral proteome. When searching, SEQUEST used a dynamic mass modification on methionine residues corresponding to oxidation (15. 99 Da) and a static mass modification on cysteinyl residues to account for alkylation by iodoacetamide (57.02 Da). Peptides passing the following filter criteria were stored as AMT tags in a Microsoft SQL Server database: 1) SEQUEST DelCn2 value (normalized Xcorr difference between the top scoring peptide and the second highest scoring peptide in each MS/MS spectrum) ≥ 0.10 and 2) SEQUEST correlation score (Xcorr) ≥ 1.6, 2.4, and 3.2 for fully tryptic peptides with 1+, 2+, and 3+ charge states, respectively, and Xcorr ≥ 4.3, and 4.7 for partially tryptic or non-tryptic protein terminal peptides with 2+, and 3+ charge states, respectively. Fully non-tryptic peptides were excluded, and a minimum peptide length of 6 amino acid residues was required. These criteria resulted in 39,744 peptides identified with an estimated false discovery rate of <2% based on a target-decoy database search [57] . The elution times for these peptides were normalized to a range of 0 to 1 using a predictive peptide LC normalized elution time (NET) model and linear regression, as previously reported [58] . A NET average and standard deviation were assigned to each identified peptide if the same peptide was observed in multiple analyses. Both calculated monoisotopic masses and observed NETs of identified peptides were included in the AMT tag database. Identified MS/MS spectra corresponding to peptides in the AMT tag database are available at the PRoteomicsIDEntification (PRIDE) database (http://www.ebi.ac.uk/pride/) under the project name A Systems Biology Approach to Emerging Respiratory Viral Diseases in the PRIDE Public Projects folder and corresponding to PRIDE Accession numbers 19855-19860.